1. Field
This disclosure is concerned generally with immunoassays and specifically with the use of immunoassays to measure components and products of the complement system of immunology and to use those measurements to determine the relative safety of biologically active products.
2. Prior Art
Immunoassays are assays based on the ability of a given antibody to complex with a very specific substance. Because of the highly specific nature of antibodies to bind with a given substance and the availability of certain "labels" that can be bound to the antibody or the substance, immunoassays can be and are used in very exquisite ways to detect very low concentrations of substances.
Some of the earlier immunoassays used radioactive isotopes as labels. These are referred to as radioimmunoassays or RIA. In another type of immunoassay, the use of isotopes is avoided by using enzymes as labels. The bonded enzymes act on substrates to generate measurable products, thus, indirectly, indicating the amount of substance bearing the enzyme label. A typical immunoassay using an enzyme label is referred to as an ELISA (enzyme linked immunosorbant assay). Since immunoassays usually require a step involving separation of reactants, it is common to immobilize at least one of the reactants on a solid support to facilitate the separation.
The complement system refers to a system of functionally linked proteins that interact with one another in a highly regulated manner to provide, ultimately, what is known generally as an immune response. The system involves what is known as the complement cascades in which various complement components (CC) interact to form various complement activation products (CAP). There are two generally recognized complement cascades, the so-called classical and alternative pathways, both ultimately leading to the formation of a complex known as the membrane attack complex (MAC), also known as C5-9. Complement components, complement activation products and the complement pathways are described in the book by A. K. Abbas et al, Cellular and Molecular Immunology, W. B. Saunders Company, 1991. See especially Chapter Thirteen, The Complement System, pp 260-82, the details of which are incorporated into this disclosure.
While there is much yet to be learned about the complement system, it is known that the complement cascades involve the presence and generation of substances known as complement activation products (such as C4a and C4b), complement complexes (e.g., C5-9) and complement components (e.g., designated C1 through C9, the three sub-components of C1 being known as C1q, C1r and C1s).
Assays for complement activation products and components have been disclosed by others. See for example, Wagner J L, and Hugli T E, Radioimmunoassay for Anaphylatoxins: A sensitive method for determining complement activation products in biological fluids. Anal Biochem 136:75-88 (1984). Those authors describe utilizing radioimmunoassay (RIA) to determine the complement breakdown products C3a, C4a, and C5a in various clinical materials (e.g. plasma samples) that may prove useful as a diagnostic tool. The assays determined the complement activation of heat-aggregated IgG at neutral pH (7.0).
See also, Tsay GC, and Stambolija L, Heat-aggregated immunoglobulin G induced anaphylatoxin generation (complement activation). Abstract, the FASEB Journal 4:A2089 (1990). This reference describes heat-aggregated IgG prepared at neutral pH (7.0) which induces anaphylatoxin generation (complement activation) in vitro by the RIA method. It was found that aggregates formed at pH 4.25 did not.
Lastly, see Ziccardi R J, and Cooper N R, Development of an immunochemical test to assess C1 inactivator function in human serum and its use for the diagnosis of hereditary angioedema. Clin Immunology and Immunpathol 15:465-471 (1980). This reference describes the diagnosis of C1-inactivator (IN) function in human serum determined with the addition of heat-aggregated IgG at pH 7.0 which activated the C1r and interacted with C1-IN in the sera. This lead to a reduction in the level of antigenically detectable C1r by radial immunodiffusion utilizing mono-specific antiserum to C1r.
We are unaware, however, of the use of immunoassays to determine the anticomplementary activity (and safety) of a biologically active therapeutic product (such as antibody preparations) intended for infusion. Details of such assays are described below.